AOAC ERP MICRO AUGUST 2018

17.5.04

( c ) Package insert. —Color comparison chart. ( d ) Blender. —High speed. ( e ) Incubator. —Maintaining 35 ° –37°C. ( f ) Centrifuge. —Capable of 1000–3000 ´ g . ( g ) Syringes. —Disposable, plastic, ca 25 mL.

AOAC Official Method 993.06 Staphylococcal Enterotoxins in Selected Foods

Polyvalent Enzyme Immunoassay Method (3M ä TECRA ä Staph Enterotoxin VIA)

( h ) Tubes. —10 mL, polypropylene. ( i ) Plastic squeeze bottle. —500 mL. ( j ) Pipets. —( 1 ) 50–240 m L, adjustable; ( 2 ) 5–50 m L, adjustable; with suitable plastic tips. ( k ) Cotton. —Absorbent. ( l ) pH Paper. —0–14 range. ( m ) Microtiter plate shaker. —Optional. ( n ) Microtiter plate reader. —Optional, but recommended; 414 ± 10 nm, single-wavelength reader; 405 and 490 nm (414 and 492 nm), dual-wavelength reader. C. Reagents ( a ) Anti-SET antibody-coated microtiter strips. —Strips of plastic wells coated with antibodies to SET (serotypes A - E) produced in sheep. ( b ) Wash solution. —Contains 1.5 g tris(hydroxymethyl)amino- methane (Tris), 6 g NaCl, 2.0 g polyoxyethylenesorbitan monolaurate (Tween 20), and 0.001 g thimerosal in 25.0 mL H 2 O. ( c ) Test suspension additive solution. —Contains 2 g Tween 20 and 0.001 g thimerosal in 6.0 mL H 2 O. ( d ) Positive control solution. —Prepare by diluting 25 m L positive control concentrate (containing SET serotype B, 0.1 g

First Action 1993 Final Action 2000

(Applicable to detection of 1.3–3.3 ng/mL staphylococcal enterotoxin in extracts prepared from selected foods [ see Table 993.06 ] containing 4–10 ng/mL staphylococcal enterotoxin. Specific toxin serotypes A to E are not differentiated.) See Table 993.06 for the results of the interlaboratory study supporting acceptance of the method. A. Principle Detection of staphylococcal enterotoxins (SETs) is based on enzyme immunoassay (EIA) using mixture of high-affinity capture antibodies to each toxin (A - E). SETs present in test extract will bind to antibody mixture that has been adsorbed onto surface of microtiter wells. Other materials in test extract are washed away. Enzyme-labeled antibodies (conjugate) specific for SETs are added, and assay results are identified by conversion of colorless substrate to green product. B. Apparatus ( a ) Strip holder. —For securing individual wells or strips of 12wells. ( b ) Strip cover. —To cover microtiter plate, use plastic film wrap or aluminum foil with plastic microtiter plate top.

Table 993.06. Interlaboratory study results for detection of Staphylococcus aureus enterotoxins in foods by visual polyvalent enzyme immunoassay method Food Enterotoxin, ng/g Reader a X b s r s R RSD r , % RSD R , % Beef/pasta 0 D 0.077 0.012 0.015 15.8 19.5 Beef/pasta 0 S 0.074 0.011 0.051 14.5 69.2 Beef/pasta 6 D 1.488 0.094 0.346 6.3 23.2 Beef/pasta 6 S 1.501 0.068 0.176 4.5 11.7 Chicken 0 D 0.075 0.009 0.019 11.5 24.9 Chicken 0 S 0.078 0.011 0.043 13.7 54.6 Chicken 4 D 0.470 0.038 0.128 8.2 27.2 Chicken 4 S 0.466 0.036 0.085 7.7 18.1 Lobster bisque 0 D 0.075 0.007 0.018 9.2 23.7 Lobster bisque 0 S 0.077 0.012 0.052 15.1 68.2 Lobster bisque 8 D 0.410 0.025 0.087 6.2 21.3 Lobster bisque 8 S 0.389 0.019 0.066 4.8 16.9 Mushrooms 0 D 0.095 0.008 0.015 8.6 16.2 Mushrooms 0 S 0.104 0.020 0.046 18.9 44.0 Mushrooms 10 D 0.435 0.031 0.099 7.0 22.7 Mushrooms 10 S 0.464 0.040 0.054 8.6 11.6 Nonfat milk 0 D 0.067 0.004 0.017 5.5 25.4 Nonfat milk 0 S 0.070 0.011 0.043 15.3 61.5 Nonfat milk 5 D 1.161 0.185 0.420 15.9 36.2 Nonfat milk 5 S 1.234 0.085 0.174 6.9 14.1 a D = Dual-wavelength microtiter plate reader; S = single-wavelength microtiter plate reader. b X = Mean absorbance value.

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